Identifying the molecular mechanisms that regulate the mammary cell differentiation hierarchy is critically important to better understand how such cell fate decisions impact mammary gland development, maturation and the initiation and progression of various subtypes of breast cancer. However, the transcriptional and gene-regulatory mechanisms that control cell lineage choices in mammary gland is not well understood, particularly in the in vivo context of the hormone-responsive pregnant mammary glands. Hence our long-term goal is to comprehensively delineate the transcriptional network of mammary glands during pregnancy. Elf5 is a key transcription factor that functions downstream of the prolactin receptor and is essential for lobuloalveolar development of the mammary gland. Using Elf5 conditional knockout animals, we have shown that Elf5-null mammary glands completely fail to initiate alveologenesis during pregnancy. In addition, Elf5-null mammary glands harbor an expanded pool of stem and luminal progenitor cells and exhibit features of Epithelial Mesenchymal Transition (EMT) - these findings further highlight the importance of Elf5 in driving cell fate choices. Although Elf5 is an essential regulator of mammary epithelial development and in particular of luminal progenitor differentiation, several key aspects of the molecular nature of the Elf5 enriched luminal progenitors remain unknown. Specifically, there is unmet need to define and mechanistically better understand how Elf5 mediates its downstream transcriptional activities in luminal progenitor cells during pregnancy. To address these knowledge gaps, we will utilize a novel transgenic mouse model that selectively expresses the Green Fluorescent Protein (GFP) reporter in the Elf5-enriched luminal progenitor cells. We will perform ChIP-exo studies using anti-Elf5 antibodies and deep sequencing to identify global Elf5 targets sites at high resolution and deep coverage in the purified luminal progenitor cells obtained from mouse pregnant mammary glands. These studies will be complemented by a bioinformatics approach to identify additional transcription factor binding sites that are enriched for and co-represented with Elf5 binding sites in regulatory regions. Collectively, these experiments will shed light on Elf5-governed transcriptional control mechanisms and signaling pathways. This work is highly innovative and significant because our proposed use of cutting edge technologies to examine fundamental transcriptional mechanisms of gene regulation will lead to new discoveries into developmental decisions of the mammary gland. Such information is not only crucial in providing important insights into the process of normal controls of mammary gland physiology during pregnancy, but will also divulge important clues on pathological conditions such as basal breast cancers, which arise from luminal progenitor cells.